By measuring the chimerism status of isolated T cell and myeloid cell subsets, we provide clinician/Investigators with valuable information both about immune and the hematopoietic engraftment of donor cells after transplanation. To assure the high quality of our results and improve the efficiency of the laboratory, during the past year, we have modified our methods for isolating T cell and myeloid cells replacing a manual method using Dynal magnetic beads with a semi-automated method using Robosep beads produced by Stem Cell Industries. Using flow cytometric methods we have confirmed the effectiveness of the new method in isolating T and myeloid cells (>95% purity), even in patients with extreme leukopenia. Working in collaboration with Dr. John Tisdale and Matthew Hsieh from NIDDK, during the past year, we have reviewed the laboratory and clinical data from a cohort of patients with severe sickle cell anemia treated by stem cell allotransplantation. These patients received a novel non-myeloablative conditioning regime which included conditioning with total body irradiation and Campath, the infusion of highly T cell depleted stem cell products, and treat with the immunosuppressive drug sirolimis posttransplant. The goal of this approach was to assure a high level of myeloid engraftment with minimal T cell engraftment. Chimerism studies in our laboratory posttransplant clearly documented the success of this approach. Most patients achieve near complete myeloid engraftment within 1 to 2 months with extremely delayed T cell engraftment. In subsequent follow-up, there have been no serious morbidity or mortality from graft versus host disease and most patients have experienced near complete disappearance of hemoglobin S from their blood with the complete disappearance of sickle cell crisis. In light of the safety of the procedure and the excellent clinical outcome, this approach represents an attractive alternative not only for patients with clinically severe sickle cell anemia but also patients with other genetic abnormalities. Indeed a modified protocol is currently being used in other protocols by Drs. Malech (NIAID) and Hickstein (NCI) to treat patients with severe chronic granulomatous disease and other life-threatening immunodeficiency syndromes. Using molecular methods to quantitate lineage specific chimerism in subpopulations of leukocytes selected by flow cytometry, we have recently recently been able to document successful engraftment in patients with severe immunodeficiency associated with a myelodysplastic syndrome. During the past year we have been asked to perform routine chimerism analysis on protocol patients (form the NCI and NHLBI) receiving two and three separate stem cell products from different donors. In some cases, this involved the infusion of human umbilical cord stem cells from two separate donors. In other instances, peripheral blood T cells and stems cells from an HLA-matched donor plus CD34 cells from another haploidentical relative were used in combination. In all cases we were technically able to quantitate the relative contributions of each donor to the total T cell and myeloid cell pool. The impact of these maneuvers on the clinical course has been variable and will be reviewed systematically by the principal investigators in the future. We have been active collaborators in a novel protocol designed within the NCI by Drs. Bishop and Hardy to retrieve surgically and expand tumor infiltrating lymphocytes for reinfusion from patients with recurrent disease after allogeneic stem cell transplant for cancer. Working closely with these investigators, we have been able to document that the tumor infiltrating lymphocytes obtained from the patient and the ex vivo expanded T cells descended from of these cells (which will be reduced into the patient) are of donor origin.